Helicase-like transcription factor (Hltf)-deletion activates Hmgb1-Rage axis and granzyme A-mediated killing of pancreatic β cells resulting in neonatal lethality

Epigenetic mechanisms are integral to pancreatic β cell function. Promoter hypermethylation of the helicase like-transcription factor (HLTF) gene—a component of the cellular DNA damage response that contributes to genome stability—has been implicated in age-associated changes in β cells. To study HLTF, we generated global and β cell-specific (β) Hltf knockout (KO) immune competent (IC) and immune deficient (ID) Rag2-/IL2- mice. IC global and β Hltf KO mice were neonatal lethal whereas ID global and β Hltf KO newborn mice had normal survival. This focused our investigation on the effects of Rag2 interruption with common gamma chain interruption on β cell function/survival. Three-way transcriptomic (RNAseq) analyses of whole pancreata from IC and ID newborn β Hltf KO and wild type (Hltf +/+) controls combined with spatially resolved transcriptomic analysis of formalin fixed paraffin embedded tissue, immunohistochemistry and laser scanning confocal microscopy showed DNA damage caused by β Hltf KO in IC mice upregulated the Hmgb1-Rage axis and a gene signature for innate immune cells. Perforin-delivered granzyme A (GzmA) activation of DNase, Nme1, showed damaged nuclear single-stranded DNA (γH2AX immunostaining). This caspase-independent method of cell death was supported by transcriptional downregulation of Serpinc1 gene that encodes a serine protease inhibitor of GzmA. Increased transcriptional availability of complement receptors C3ar1 and C5ar1 likely invited crosstalk with Hmgb1 to amplify inflammation. This study explores the complex dialog between β cells and immune cells during development. It has implications for the initiation of type I diabetes in utero when altered gene expression that compromises genome stability invokes a localized inflammatory response.


Introduction
A balance between genome stability and genome diversification is required for survival of the species.Genome stability is the ability to preserve and faithfully transmit genetic material from cell to cell which includes Helicase-like transcription factor (HLTF) in error-free replication of DNA and the repair of damaged DNA [1].Genomic instability is a hallmark of most cancers [2], and epigenetically silenced HLTF occurs frequently in colorectal cancer [3].Initially, HLTF was identified as an E3 ubiquitin ligase that polyubiquitinylated PCNA and stimulated error-free post replication repair [4].Subsequently, HLTF was shown to mediate replication stress via its fork reversal activity [5] and removal of damage-containing oligonucleotides to facilitate nucleotide excision repair [6].
DNA damage resulting from replication stress can result in increased DNA damage repair.Conversely, unrepaired DNA damage promotes cell death by apoptosis.DNA fragmentation in the later phase of apoptosis [7] can lead to the aberrant release of DNA fragments in the cytoplasm and trigger innate immune responses [8].High mobility group box 1 (Hmgb1), a chromatin binding non-histone nuclear protein, the second most abundant protein in the nucleus, is passively released into the extracellular microenvironment during apoptosis [9].Once outside the cell, Hmgb1, a damage associated molecular pattern molecule (DAMP), engages the pattern recognition receptor for advanced glycation end products (RAGE), to form a proinflammatory axis and alerts the innate immune system to excessive deregulated cell death [10].Excessive activation of the innate immune signaling can lead to the development of autoimmunity [11].
In this regard, pancreatic β cells have been implicated in their own demise [12].Type 1 diabetes (T1D) has long been described as an autoimmune disease in which the β cells are mistakenly destroyed by immune cells.However, it is possible that an epigenetic event in β cells could alter their dialog with islet-resident immune cells.Macrophages that initially function in a mediator role removing catabolic products could now respond to DAMPs leading to β cell destruction.Two lines of evidence support this concept.The first is the long-standing fact that genetic changes only account for ~50% of T1D [13]; and the second is compromised immune homeostasis in the islet microenvironment implicates immune surveillance in the pathology of T1D [14].Therefore, we examined the role of Hltf, a known epigenetic target, in β cell death/ survival using various KO models including β cell-specific KO mice and found a role of Hltf in preventing DNA damage thereby promoting β cell survival.
Primary and secondary antibodies used with FFPE tissue sections in immunohistochemistry (IHC-P) and immunofluorescence (IF) are listed in Table

Hltf-deleted mouse models and controls
IC global Hltf KO mice were developed in collaboration with genOway (Lyon, France) as previously described [15,16].Mice with either the Hltf-deletion or floxed Hltf-gene were bred to the recombinase activating gene 2 (Rag2)/common gamma (IL2rg) double knockout mice [17], thereby generating ID Hltf KO or Hltf-fl/fl mice.Mice with a floxed Hltf-gene that were either IC or ID were bred to Rip-Cre TG mice (The Jackson Laboratory, Stock No. 003573) thereby deleting Hltf selectively in β cells (β Hltf KO) All mice are in sentinel-monitored, rodent housing in the Laboratory Animal Resource Center (LARC) at Texas Tech University Health Sciences Center (TTUHSC).Additionally, mice on the Rag2)/IL2rg double knockout background are in bioBubble™-husbandry conditions in the LARC.Pathogen free mice were able to access food and water ad libitum.All studies were in accord with the NIH Guidelines for the Care and Use of Laboratory Animals, as reviewed and approved by the Animal Care and Use Committee at TTUHSC [NIH Assurance of Compliance A3056-01; USDA Certification 74-R-0050, Customer 1481, S1 Checklist].TTUHSC's IACUC specifically approved this study.Pain and suffering were always minimal.We previously reported the surgical removal of unborn pups and their placentae [17] from term pregnant females (n = 2).Each female received an IP injection of a Ketamine/Xylazine cocktail at 100 microliters per 20 g body weight.The cocktail consisted of 87.5 mg/kg Ketamine and 12.5 mg/kg Xylazine.Results from studies with the placentae were already published [17], whereas histology of pancreata from pups is reported here.Previously pregnant females were euthanized by drug overdose followed by cervical dislocation.

Serum collection from newborn mice
Postprandial newborn mice are unable to thermoregulate, and were placed on infrared warming pads (37˚C) to avoid the negative effects of hypothermia on blood glucose prior to decapitation with surgical scissors.Blood glucose in trunk blood was measured immediately in all members of each litter with the exception of pups that were already dead.Low (�15 mg/dL) blood sugar in global and β Hltf KO mice affected anywhere from one pup in the litter to the entire litter.We used the MiniCollect1 capillary blood collection system to collect trunk blood.Serum was removed from clotted blood after centrifugation.Serum samples (5-30 μl) were stored frozen (-20˚C) until use in an ultrasensitive insulin test.Tails from pups were used for genotyping and gender authentication.

Mouse ultrasensitive insulin ELISA
The Mouse Ultrasensitive Insulin ELISA (Alpco) quantified the concentration of insulin protein from mouse I and mouse II proinsulin genes according to the manufacturer's instructions.There was no cross reactivity with mouse C-peptide 1 or 2, or mouse IGF 1 or 2. Because 25 μl of serum was required for hypoinsulinemic samples, it was necessary to pool serum samples in a gender specific manner as shown in Table 3.

Analysis of pancreatic tissue
Abdominal segments of IC Hltf +/+ (control) and global Hltf KO E18.5 mice (N = 8/group) were formalin fixed overnight at 4˚C, paraffin embedded, and serially sectioned (4 μm).Tissue sections were either stained with Hematoxylin and Eosin (H&E), or immunostained for insulin.Diaminobenzidine was the chromogen.Abdominal segments of newborn IC pups were infused with formalin-based fixative (n = 12 each) for global Hltf-KO with low (�15 mg/dL) blood sugar, Hltf +/+ control, and β Hltf KO with low blood sugar.In companion experiments, abdominal segments of ID new born pups of the same three genotypes were infused with formalin-based fixative.All tissues were fixed overnight at 4˚C, paraffin embedded, and serially sectioned (4 μm).
Immunocytochemistry (Table 1) for laser scanning confocal microscopy was performed with serial sections from the above described groups of newborn pups with HIER, aldehyde quench (50 mM NH 4 Cl in PBS), and ProLong Gold DAPI.

TUNEL assay
Apoptosis was determined using the DeadEnd TM Fluorometric TUNEL assay with slides from the samples used for quantification of insulin expression (above) according to the manufacturer's instructions.Negative controls included sections incubated without the TdT enzyme, and were devoid of a positive reaction.For quantification, the area of TUNEL positive cells was determined using particle analysis (internal function of Image J) in pixels 2 for each image.Contrast enhancement expanded the dynamic range of images, and color threshold was set at a constant value, which only selected the positive staining areas.To control for tissue size, the total area of TUNEL positive cells was normalized to the total tissue area.

Statistical analysis
All values are expressed as the mean ± standard error of the mean (SEM) of n independent experiments.With the exception of RNA-seq and spatial transcriptomics, all data analyses were conducted with GraphPad Prism version 9.1.1software.For multiple comparisons, we performed a one-way analysis of variance (ANOVA) with an appropriate post hoc test as described for each experiment, p<0.05 was significant.

Pancreatic transcriptome
Because pancreata are ribonuclease-rich [19], trunks of decapitated newborn mice were perfused in situ with RNAlater by insertion of a 20g-1-inch needle attached to a 5 ml syringe into the abdomen [20] via the crural (posterior) attachment of the diaphragm [21].RNA stabilization occurred concomitant with the initial stretching of the pancreas.Pancreata were stored in RNA later at -70C until total RNA was isolated.RNA integrity and purity were assessed (Agilent Bioanalyzer) for 10 samples, i.e.GEX slide.Four capture areas (6.5 x 6.5 mm each) inside fiducial frames measure 8 x 8 mm.Each capture area contains 5,000 gene expression spots (55 μm in diameter) spaced with a distance of 100 μm between the centers of each spot and captures gene expression data for 1-10 cells.Visium for FFPE uses RNA-templated ligation (RTL) probes targeting the whole transcriptome.The assay does not capture transcripts directly, but captures probes via a capture sequence, e.g.poly-A for Visium for FFPE probes.Each gene expression spot has primers with a unique spatial barcode Probes are designed against the entire mouse genome, each with primers that include Illumina TruSeq Read 1 (partial read 1 sequencing primer), 16 nt spatial barcode (all primers in a specific spot share the same spatial barcode), 12 nt unique molecular identifier (UMI), and 30 nt poly(dT) sequence (captures ligation product).Spatially barcoded, ligated products were released from the slide, and harvested for qPCR with KAPA SYBR Fast qPCR master mix.The threshold for determining the Cq value for each sample was set along the exponential phase of the amplification plot at ~25% of the peak fluorescence value with QuantStudio 12 K Flex real-time PCR system (ThermoFisher Scientific).Sample index sets were selected to distinguish each of the 4 samples in a multiplexed sequencing run.Samples were amplified using Ilumina-compatible indexing primers, cleaned up with SPRIselect reagent, and bi-directionally sequenced.
Mouse probe set.Visium Mouse Transcriptome Probe Set v1.0 contains 20,551 gene ids targeted by 20,873 probes.Gene ids (1,086, 5.3%) targeted by 1,110 probes were excluded by default due to predicted off-target activity to a different gene.As a result, 19,465 gene_ids (targeted by 19,763 probes) were present in the final filtered output.During data analysis, read 2 sequences were mapped against the reference mouse genome C57BL/6J (GRCm38/mm10) and read 1 sequences were used for UMI filtering to obtain spatial information.
Sequencing.Illumina NovaSeq at GenWiz (Azenta Life Sciences, South Plainfield, NJ).Unique dual indexing-unique identifiers on both ends of the sample-allows for an increase in the number of samples sequenced per run and reduces per-sample cost compared to other indexing strategies.Sequencing depth was a minimum of 50k read pairs per spot covered with tissue.This was calculated by estimating the percent of capture area covered by the tissue section based upon the H&E brightfield image.Actual values are provided in Table 5.
Bioinformatics analysis.Bioinformatic analysis utilized the Visium Spatial Gene Expression Software Suite that includes Space Ranger and Loupe Browser.Space Ranger has three pipelines for FFPE data analysis.
Spaceranger mkfastq demultiplexed the Illumina sequencer's base call files (BCLs) for each flow cell directory into FASTQ files.Spaceranger count combined a brightfield microscope slide image and FASTQ files from spaceranger mkfastq and performed alignment, tissue detection, fiducial detection, barcode/UMI counting, and prepared a full resolution slide image for visualization in Loupe Browser.The pipeline used the Visium spatial barcodes to generate feature-spot matrices, determine clusters, and perform gene expression analyses.The pipeline uses a probe aligner algorithm for FFPE tissues.Spaceranger aggr used the output of multiple runs of spaceranger count from related samples and aggregated their input, normalizing those runs to the same sequencing depth, and then recomputed the feature-barcode matrices and the analysis on the combined data.The aggr pipeline combined data from multiple samples into an experiment-wide feature-barcode matrix and analysis.Loupe Browser was used to interrogate significant genes, characterize and refine gene clusters, and perform differential expression analyses.

Perinatal lethal phenotype
Phenotypic analysis of global Hltf KO mice was challenging because the gene deletion caused perinatal death.Survivors have contributed to our understanding of Hltf gene function in cancer [22,23]; however, until now, the exact mechanism responsible for perinatal death has remained obscure.Although a single gene deletion can alter more than one physiological system, studies on brain [15], heart [16] and placenta [17] only served to eliminate Hltf-deletion from these organs as directly responsible for perinatal lethality.In contrast, morphological evaluation of pancreata on mouse embryonic (E) day 18.5, indicated the mechanism underlying the complex phenotype was established in utero.Microscopic observation (Fig 1 ) shows typical pancreatic organization with normal acinar and ductal tissue and insulin-positive islets containing β cells in Hltf +/+ (control) and IC global Hltf KO mice.However, upon closer inspection, the islets in control tissue are more robust compared to the islets in IC global Hltf KO mice that appear smaller with distorted histomorphology (Fig 1C -1F   blood sugar commensurate with the survival statistics.Importantly, of the 57% of newborn mice with low blood sugar (Fig 2C ), males were affected more frequently (3:2 ratio) than females.This finding is consistent with the unexplained fact that diabetes is more frequent in men than women.rIPCre transgenic mice were used to generate β Hltf KO mice in order to eliminate potential global Hltf-deletion effects-on gut hormones, glucagon secretion, nutrient-sensing neurons in glucose homeostasis, and glucose uptake by liver, adipose and muscle -that would alter glucose metabolism.

Pancreatic transcriptome
To understand Hltf-deletion effects on β cell development and resultant changes in gene expression, we performed three-way transcriptomic (RNAseq) analyses of whole pancreata from IC β Hltf KO, ID β Hltf KO and IC control (Hltf +/+) newborn mice.The microenvironment (interstitial matrix, peri-islet basement membrane, and microvascular cells), as it was potentially altered by β cell-specific Hltf gene-deletion, was an integral component of the experiment.Using the iPathwayGuide analysis tool, RNA-seq data showed transcription of two genes most exclusively expressed in β cells-nonallelic insulin 2 (Ins2) and insulin 1 (Ins1) genes-as well as Neurogenin3 (Neurog3), sex-determining region Y (SRY)-related high mobility group (HMG) box (SOX) transcription factor 4 (Sox4), pancreatic polypeptide (Ppy)lineage β cells and pancreatic and duodenal homeobox 1 (Pdx1) were transcriptionally downregulated in IC β Hltf KO mice.These data confirmed that Hltf-deletion in the presence of an intact immune system is associated with β cell loss during development.These findings are consistent with data showing reduced insulin hormone in whole tissue and in the systemic circulation.Additional findings were exclusionary.Hltf-deletion throughout embryonic development produced no altered molecular signature (Ngn 3, Pou5f, and Mycl1) suggesting the progenitor cell state was maintained.There was no effect of β cell-specific Hltf-deletion on transcription of glucagon (α cells) or somatostatin (δ cells) genes.
To accommodate the long expanses of interconnected islets located along large blood vessels in the neonatal pancreas [26] and to preserve neonatal intra-islet architecture, spatial transcriptomics-which incorporates unbiased total mRNA analysis in intact FFPE sections of abdominal segments from IC and ID β Hltf KO mice in a morphological context-globally distinguished 22

Murine Hltf is alternatively spliced
Post-transcriptional processing yields a full-length message isoform (4955-bp; exons 1-25) and a 3´-truncated isoform (3059-bp; exons 1-21 with exon 21 extended via an intron retention event) in mouse brain [15] and heart [16].Term placenta exclusively expresses the short isoform [17].The full-length mRNA encodes a full-length protein with a DNA repair domain.The short mRNA encodes a nearly identical truncated protein that lacks the DNA repair domain.Hltf-deletion of the full-length splice variant, confirmed by RNAseq, in pancreas precludes the protein's DNA-damage response from benefiting β cells.DNA damage from double stranded breaks is quickly followed by phosphorylation of Ser-139 of the histone variant H2AX.Foci of the newly phosphorylated protein, known as γH2AX, is a hallmark of DNA damage associated with a germline HLTF mutation in familial myelodysplastic syndromes

Discussion
Autoimmune destruction of pancreatic β cells is poorly understood, in part, because it is unclear how the β cells and immune cells interact to initiate or perpetuate the process.In this  activated Hmgb1-Rage axis mediated increased β cell visibility to immune surveillance.We know from the literature that GzmA activates a caspase-independent cell death pathway with morphological features of apoptosis (nuclear fragmentation) via single-stranded DNA damage measurable with TUNEL.Perforin-delivered GzmA to target cell cytoplasm where it activates the endonuclease Nme1 that works in concert with the exonuclease Trex1 to nick DNA [28].GzmA also traffics to and concentrates in the nucleus.GzmA disrupts the nuclear envelope by cleaving lamins.GzmA further interferes with DNA repair by cleaving and inactivating Parp-1, an ARP-ribosyl transferase enzyme [29], that functions as an early sensor of both single and double stranded DNA damage.GzmA cleaves Parp-1 to separate its DNA binding domain from its catalytic domain.The complete loss of Hltf-Parp1 interaction [30] in the progression This caspase-independent method of cell death was supported by transcriptional downregulation of the Serpinc1 gene that encodes a serine protease inhibitor of GzmA.No other inhibitors of granzymes have been identified other than this one presumably because GzmAthe most abundant serine protease in killer cell cytoplasmic granules-may require rapid removal from the extracellular milieu.A recent study on the expression of complement receptors, C3ar1 and C5ar1, on human and mouse β cells, indicated they are positive regulators of cell function [31].This may be true for ID β Hltf KO cells.However, increased transcriptional availability of the complement receptors C3ar1 and C5ar1 likely invited crosstalk with Hmgb1 to amplify inflammation in IC β Hltf KO cells [32] given the evolutionary conservation of the complement system [14].Our study is not precisely analogous to neonatal diabetes because IC β Hltf KO mice do not have low-birth weights [17] compared to controls (Hltf +/+).However, like neonatal diabetes [33] the study does show that immune attack can start before birth and progress rapidly to complete destruction of insulin producing β cells.Our findings are otherwise compatible with two studies that identified Hltf in the β cell transcriptome [34] and proteome [35] implicating Hltf in pancreatic β cell function.

Conclusions
HLTF is well known for its role in post-replication repair, and more recently for efficient nucleotide excision repair.To our knowledge, ours is the first in vivo experimental system to evaluate the loss of Hltf-facilitated DNA repair in the presence and the absence of the immune system.The results show conclusively that the innate immune system compromises DNAdamage repair and survival when Hltf is deleted from pancreatic β cells during development.In as much as disturbances in epigenetics mechanisms can result in developmental dysfunction and disease processes, epigenetic reprogramming has corrective potential.
). Reduced insulin expression was evident in pancreata from IC global Hltf KO at both low (Fig 1B) and high (Fig 1F) magnifications compared with controls (Fig 1A and 1D).

Newborn
IC global Hltf KO mice and their littermate controls are pink in color and display a sucking reflex immediately after birth.Despite the presence of milk in their stomachs, 75% of newborn IC global Hltf KO mice lose their surface righting reflex, and display central cyanosis (Fig 2A), consistent with hypoglycemia (Fig 2B).Reevaluation of blood sugar data from IC global Hltf KO mice (Fig 2C) shows the percentage of mice with low or marginal (20-25 mg/ dL) blood sugar levels comprised 73% of the population of IC global Hltf KO mice.With the minimum threshold set at 26 mg/dL, 27% of the total population of newborns achieved normal Fig 2D shows PCR validation of the genotype.Because rIPCre mice alone display glucose intolerance as early as 6 weeks of age [24, 25], it was imperative to include all the appropriate controls to show Hltf fl/fl as well as rIPCre Hltf +/+ mice have normal blood sugar at birth compared with Hltf +/+ control pups (Fig 2B).IC global and β Hltf KO mice share the same dramatic reduction in circulating levels of blood glucose and the same perinatal lethal phenotype indicating it is solely attributable to alterations in islet β cells (Fig 2B).Potential confounding effects of the innate immune system on pancreatic development/ function were eliminated when the Hltf-deletion line was bred into the recombinase activating gene 2 (Rag2)/common gamma (IL2rg) double knockout background.Unexpectedly, the perinatal lethal phenotype was eliminated, i.e. newborn ID global and β Hltf KO mice were born euglycemic (Fig 2B) with survival rates equivalent to IC Hltf +/+ controls.Because IC global and β Hltf KO mice share the characteristic of low blood sugar levels compared to controls, we measured their non-fasting serum insulin levels and quantified insulin expression in their pancreata.As shown in Fig 3, serum insulin levels for IC global and β Hltf KO mice are reduced

Fig 2 .
Fig 2. Composite phenotype.(A) Appearance of postprandial newborn IC Hltf KO mice.Note milk-filled stomach (milk spot) in each pup, which indicates they were born alive and capable of suckling.(B) ANOVA analysis (p<0.0001) and Tukey's multiple comparisons test of blood sugar shows reduced blood sugar levels are identical (p = 0.9999) for IC global and β Hltf KO mice.Findings confirm negligible effects of the rIPCre transgene on blood sugar levels in newborn mice.Blood sugar levels for ID control and global Hltf KO pancreata do not differ (p = 0.9261) from each other compared to β Hltf KO pancreata that differ (p<0.0001)from the other two values but are comparable (p = 0.6520) to IC Hltf +/+ (control) mice.Values are mean ± SEM, and values with the same letter designation are not significantly different (p>0.05).(C) Data from IC global Hltf KO mice show the percentage of mice with low blood sugar is nearly identical to the rate of neonatal lethality.(D) PCR validation of genotypes with amplicons of the expected sizes shown for fl/+ genotype (Lane 1), fl/fl genotype (Lane 2), ϕX174 DNA/Hae III molecular size markers (Lane 3) and rIPCre transgene (Lane 4).https://doi.org/10.1371/journal.pone.0286109.g002 graph-based clusters (Fig 4A).Next, tissue transcriptomics and gene deconvolution were used to quantify differential gene expression in pancreata from IC (cluster 18) vs ID (cluster 16) samples (Fig 4B).Mapping insulin 1 and insulin 2 gene expression in islets from IC and ID β Hltf KO mice (Fig 4C) confirmed reduced insulin gene expression in IC β Hltf KO mice.

Fig 4 .
Fig 4. Exploration of the tissue architecture of IC and ID β HLTF KO pancreata.(A) Two-dimensional batch-corrected t-distributed stochastic neighbor embedding (t-SNE) visualization of the UMI counts from the entire IC vs ID dataset.(B) Pancreata clusters 16 and 18 are shown in t-SNE space.(C) Pancreatic genes insulin 1 (Ins1) and insulin 2 (Ins2) illustrated differential gene expression in IC vs ID β Hltf KO mice in t-SNE plots of clusters 16 and 18.The unique expression of immune cell markers in IC vs ID tissue is elaborated in t-SNE plots of these clusters.Slamf6 and Il2rb were added to previously identified markers for NK cells (GzmA, Klrb1b).Five markers for B cells (Pax5, Blk, Fcmr, Fcrla, Tnfrasf9) and three markers for activation of innate immunity (Bpifb1, Serpinb3a, Defb36) were unique to cluster 18. https://doi.org/10.1371/journal.pone.0286109.g004

Fig 5 .
Fig 5. Differential γH2Ax pan-staining and TUNEL assay.Abundant γH2Ax in β cells from IC β Hltf KO mice (A) compared to minimal immunostaining in β cells from ID β Hltf KO mice (B).Two-types of γH2Ax pan-staining are evident.β cells from IC β Hltf KO mice have apoptotic rings and the β cells from ID β Hltf KO mice have limited pan-nuclear staining of the entire nucleus.Results from the terminal deoxynuceotidyl transferase dUTP nick-end labeling (TUNEL) assay (C), which detects β cell death-associated DNA fragmentation (3'-OH termini), indicates the amount of DNA damage is more than the targeted β cells can efficiently repair when the animals are IC.Cell-death in IC Hltf +/+ controls and ID β Hltf KO mice was negligible.A positive mouse testis control (D) was included because apoptosis is an important component of normal spermatogenesis.https://doi.org/10.1371/journal.pone.0286109.g005

Fig 6 .Fig 7 .
Fig 6.Nuclear morphology and DNA damage in response to β Hltf KO.The confocal image analysis was driven by the desire to visualize the integrity of nuclear DNA (DAPI, blue) in the context of the F-actin cytoskeleton (phalloidin, green) using insulin (red) as a definitive β cell identifier.DAPI binds the ATrich regions of double-stranded DNA.Binding is accompanied by a 20-fold enrichment in fluorescence that is directly proportional to the amount of DNA as shown for Hltf +/+ (A) and ID β Hltf KO (B).In contrast, when the cell membrane is compromised by apoptosis, more DAPI enters the cells and stains a stronger blue color as shown for IC β Hltf KO (C).Chromosome condensation and DNA fragmentation enhances the visual identification of apoptotic cells stained with DAPI.Cell shrinkage occurs as a result of the serine/threonine kinase rearranging the cytoskeleton as visualized by staining for actin in merged immunofluorescence images.Colocalization (orange) of insulin (red) with the actin cytoskeleton (green) is shown for Hltf +/+ (A) and ID β Hltf KO (B).Actin is cleaved during late stage apoptosis thus actin immunostaining is negligible for IC β Hltf KO (C).For data presentation, the maximal projection confocal images obtained from a z stack (500 nm slice) using a 60x objective (oil) are shown.All images were obtained with transmitted light and excitation at 405 nm (DAPI), 488 (F-actin) and 647 nm (insulin).https://doi.org/10.1371/journal.pone.0286109.g006

Spatial transcriptomics Work flow.
3 from β Hltf KO IC mice with low blood sugar, 3 from Hltf +/+ controls, and 4 from β Hltf KO ID mice.cDNA was generated from Ribo-Zero Plus rRNA-depleted samples and subjected to Illumina library preparation.Libraries were sequenced utilizing Illumina sequencing technology.Paired-end 100 nucleotide reads were aligned to reference mouse genome C57BL/6J (GRCm38/mm10) and analyzed using the platform provided by DNAnexus, Inc. (Mountain View, CA) to generate three-way transcriptomic Five basic steps were necessary to implement spatial transcriptomics technology.Step 1, placement of FFPE tissue (abdominal segments) on capture areas of a Visium gene (RNAseq) analyses of whole pancreata from β Hltf KO IC and ID newborn mice, and wild type (Hltf +/+) controls.The analysis included alternative splicing analysis in control (Hltf +/+) pancreata.The power in detecting alternative splicing was dramatically increased by pairedend sequencing relative to single-end sequencing.FPKM (fragments per kilobase of transcript per million mapped reads) were mapped against mm10 with Tophat (V1.3.3) to obtain.bammappingfiles that were input into Cufflinks for transcript assembly.Cuffdiff (V 1.3.0),part of the Cufflinks package, used the alignment reads for rigorous statistical comparison of the three genotypes.The depth of sequencing (Table4) was a minimum of 20 million sequencing reads per sample [90% Power, 5% significance level: 91+/-4% of all annotated genes are sequenced at a frequency of 0.1 times/10 3 bases X 3 x 10 9 bases/sequencing read x 3 samples = 9 x10 4 reads/gene].All RNA-seq data were deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE137060.(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE137060)).Data were imported into iPathwayGuide (Advaita Corporation) a next-generation pathway analysis tool.Standard enrichment parameters (log2 fold change, log2 FC = 0.6, p<0.05) were used.

Table 4 . Sample quality control and RNAseq outcomes.
Step 2, H&E staining followed by brightfield microscopic imaging with ZEISS Axioscan 7 high-performance slide scanner (White Plains, NY).Step 3, permeabilization of tissue and construction of barcoded libraries with a final sample index PCR all according to the manufacturer's instructions.Step 4, NGS short-read sequencing (Illumina NovaSeq) of barcoded libraries by Genewiz (Azenta US, Inc, South Plainfield, NJ).Step 5, data analysis of tissue images and sequencing files in FASTQ format with Space Ranger run on Ubuntu 22.04 LTS-Thelio Mira-b3 by System76, Inc. (Denver, CO).The space ranger aggr pipeline was used to aggregate data from replicate samples and from samples from the different biological conditions (IC, ID).Loupe browser visualization software was accessed in a desktop application via Windows (Dell Optiplex 990).FFPE sections.Abdominal tissue sections (5 μm) from IC and ID β Hltf KO newborn mice were processed with the RNeasy FFPE kit for DV200 analysis.Replicate sections from IC and ID β Hltf KO newborn mice were placed within fiducial frames of capture areas A,B and C,D respectively, on Visium GEX slide V11D13-089-A1.10X Genomics best practices guide helped to maintain tissue adhesion and RNA integrity before and after sectioning.